Tobacco-specific N-nitrosamines (TSNA) are formed from the alkaloids during tobacco curing and smoking. The total amount of the carcinogenic TSNA in smokeless tobacco (1-80 ppm) or in smoke (1-5 Mug/cigarette) exceeds that of nitrosamines in other nonoccupational environments by at least two orders of magnitude. Based on preliminary data, it appears most likely that TSNA are also formed endogenously from nitrite-nitrate or nitrogen oxides and the alkaloids. In this study, in vivo formation of TSNA will be investigated with two approaches. First, we will establish the correlation between the in vivo formation of TSNA and N-nitrosoproline (NPRO), a noncarcinogenic nitrosamine which is known to be formed endogenously in man and is excreted exclusively in urine. For the assay of NPRO in the urine of smokers and nonsmokers, we will develop a RIA method and apply it to 10 smokers and 10 nonsmokers on a controlled diet and to groups of 30 light, moderate, and heavy cigarette smokers each, to 30 nonsmokers and to passive smokers on a regular diet. The urine will also be analyzed by RIA, for nicotine and cotinine and (by color reactions in an autoanalyzer) for nitrite, nitrate, thiocyanate, and ascorbic acid. The data will be evaluated statistically for correlations. For the second approach, we will synthesize 5-(3-pyridyl)proline (PY-PRO) and N-nitroso-5-(3-pyridyl)proline in order to study the endogenous formation of PY-NPRO in mice and rats as well as in hamsters exposed to cigarette smoke. In vitro studies with human saliva and blood will parallel these experiments. If PY-NPRO is found to be noncarcinogenic in mice and rats and if this compound will be excreted exclusively in the urine of rats and hamsters, it could serve as an ideal indicator for the extent of the endogenous formation of the carcinogenic TSNA. At present, we do not know whether TSNA are formed endogenously in chewers, snuff dippers or smokers. One cannot distinguish between uptake and in vivo formation of TSNA because these nitrosamines give the same metabolites as the parent alkaloids. Information on the endogenous formation of TSNA is of great public health interest and could lead to measures for the inhibition of their in vivo formation by natural food components such as ascorbic acid.